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研究生:邱哲琳
研究生(外文):Che-Lin Chiu
論文名稱:核苷;誘導人類肝癌細胞株HepG2與Hep3B細胞程式凋亡之影響
論文名稱(外文):Effects of nucleosides on the induction of apoptosis in HepG2 and Hep3B cells.
指導教授:楊素卿楊素卿引用關係
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:保健營養學系
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:76
中文關鍵詞:HepG2Hep3B細胞程式凋亡核苷
外文關鍵詞:HepG2Hep3Bapoptosisnucleosides
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本研究針對核苷誘導人類肝癌細胞株HepG2和Hep3B 細胞程式凋亡之影響進行探討。實驗以不添加(控制組)或添加核苷之培養液進行HepG2和Hep3B之培養。本實驗使用六種核苷包括:Inosine(I)、Cytidine(C)、Uridine(U)、Thymidine(T)、Adenosine(A)和Guanosine(G)。在37℃、5%CO2環境下培養,3、6、12、18和24小時後,收集細胞內、外液進行分析。由細胞存活率結果可知,由HepG2方面,於培養液中添加30mM I、30mM C、30mM U、30mM T、0.5mM A和1mM G,於培養後12和24小時,會造成細胞存活率顯著較控制組低(p<0.05);而Hep3B方面,則是添加30mM I、30mM C、30mM U、30mM T後,培養12和24小時,造成細胞存活率顯著較控制組低(p<0.05),0.1mM A和0.2mM G則無差異。細胞程式凋亡現象觀察之結果,在HepG2細胞週期第12小時,C、U、T、A與G組別皆可使sub G0期顯著較控制組高(p<0.05),而Hep3B在細胞週期第24小時,C、U、T與A組別皆可使sub G0期顯著較控制組高(p<0.05)。Caspase-3活性結果方面,HepG2在第6小時只有T組顯著較控制組高(p<0.05),而Hep3B在第6小時,I、T、A與G組皆可使Caspase-3活性顯著高於控制組(p<0.05)。P53與p21蛋白質在HepG2中皆可測得,但在Hep3B中皆無法測得。細胞程式凋亡作用機轉結果,由細胞外液水溶性Fas-Ligand結果可知,HepG2在第18小時發現,C、U、T與A組別皆顯著較控制組高(p<0.05),而Hep3B在第12小時,I、T、A和G組皆可使水溶性Fas-Ligand濃度顯著較控制組高。另外,由細胞外液TNF-α結果與細胞內cytochrome c蛋白質結果可知,在HepG2與Hep3B細胞中皆無法測得TNF-α與cytochrome c蛋白質。由本研究結果可知,嘌呤核苷與嘧啶核苷雖然可以顯著降低人類肝癌細胞株HepG2與Hep3B之細胞存活率,但是由細胞程式凋亡現象及作用機轉之結果,無法證實其細胞存活率降低是由於促進細胞程式凋亡所引起。
關鍵字:HepG2、Hep3B、細胞程式凋亡、核苷
The purpose of this study is to investigate whether nucleosides can induce the apoptosis in human hepatoma HepG2 and Hep3B cells or not. The nucleosides which were used in this study included inosine (I), cytidine(C), uridine (U), thymidine (T), adenosine(A) and guanosine(G). Cells were incubated by the mediums with or without nucleosides at 37℃ in a 5% CO2 humidified atmosphere. Then, the cells and mediums were collected and analyzed after incubation. It was found that the cell viability was significantly decreased in HepG2 cells which were treated with 30mM I, 30mM C, 30mM U, 30mM T, 0.5mM A and 1mM G after 12 and 24 hours incubation (p<0.05). On the other hand, the reduction of the cell viability in Hep3B which were incubated with 30mM I、30mM C、30mM U and 30mM T after 12 and 24 hours were also observed(p<0.05). But the cell viability in Hep3B, which was incubated with 0.1mM A and 0.2mM G after 12 and 24 hours, were no different. About the apoptotic phenomenon, the cell percentages of Sub G0 phase were significantly increased in HepG2 cells, which were treated with nucleosides such as C, U, T, A and G (p<0.05). And, the Hep3B cells in sub Go phase were also raised, when the medium were added with C, U, T and A. Furthermore, the caspase-3 activity was increased when HepG2 cells were incubated with T (p<0.05). In the Hep3B cells, the caspase-3 activities were increased when the mediums contained I, T, A and G (p<0.05). The expressions of p53 and p21 were detected in HepG2 cells. However, there was no expression of p53 and p21 in Hep3B cells. To investigate the mechanism of apoptosis induced by nucleosides, it was found that the content of soluble Fas ligand (sFas L) was increased in HepG2 cells following C, U, T and A treatment (p<0.05). Moreover, the Hep3B cells was also increased in sFas L contents when mediums contained I, T, A and G (p<0.05). But, there was no detection of TNF-α and cytochrome C in HepG2 and Hep3B cells. In conclusion, although purine and pyrimidine nucleosides could reduce the cell viability, that could not confirm reduction of the cell viability by apoptosis in results of apoptotic phenomenon and mechanism of apoptosis.
Key words : HepG2、Hep3B、apoptosis、nucleosides
目錄
中文摘要 I
英文摘要 III
致謝 V
表目次 X
圖目次 XI
第一章 緒論 1
第二章 文獻回顧 3
第一節 肝癌 3
第二節 細胞程式凋亡(Apoptosis) 5
第三節 細胞週期 8
第四節 p53蛋白質 10
(一) P53 10
(二) P53與細胞週期之調控 10
(三) P53與apoptosis之關係 11
第五節 核苷 12
(一) 核苷之形成 12
(二) 核苷的吸收及進入細胞的途徑 12
第六節 核苷誘導癌細胞apoptosis之相關研究 14
第三章 實驗試劑、儀器與流程 16
第一節 實驗使用之試劑 16
(一) 細胞培養使用之試劑 16
(二) 細胞存活率之試劑 16
(三) Apoptosis之試劑 17
(四) 蛋白質定量與分析之試劑 17
第二節 實驗使用之儀器 19
第三節 肝癌細胞之培養 20
第四節 實驗流程 21
第四章 細胞存活率之探討 22
第一節 細胞存活率之測量方法 22
第二節 細胞存活率之結果 23
第五章 Apoptosis之現象觀察 30
第一節 細胞週期之分析 30
(一) 細胞週期之分析方法 30
(二) 細胞週期之分析結果 32
第二節 Caspase-3活性之分析 40
(一) Caspase-3活性分析之方法 40
(二) Caspase-3活性分析之結果 41
第三節 P53、p21與procaspase-3蛋白質之表現 45
(一) 細胞蛋白質含量之分析 45
(二) 十二基硫酸鈉電泳法 45
(三) 西方點墨法 47
(四) 結果 48
第六章 Apoptosis作用機轉之探討 53
第一節 Soluble Fas-Ligand (sFas- Ligand)濃度之分析 53
(一) Soluble Fas-Ligand (sFas- Ligand)濃度分析之方法 53
(二) Soluble Fas-Ligand (sFas- Ligand)濃度分析之結果 55
第二節 Tumor Necrosis Factor-α (TNF-α)濃度之分析 59
(一) TNF-α濃度分析之方法 59
(二) TNF-α濃度分析之結果 60
第三節 Cytochrome c蛋白質之表現 61
(一) Cytochrome c蛋白質分析之方法 61
(二) Cytochrome c蛋白質分析之結果 61
第七章 綜合討論 63
第八章 總結 75
參考文獻 76
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